Preliminary Study On The Effect Of Homocysteine On Autophagy Of THP-1 Macrophages

Objective:To explore the effect of homocysteine on autophagy of THP-1-derived macrophages and its related molecular mechanism,so as to provide new ideas and strategies for exploring new targets for the prevention and treatment of atherosclerosis.Methods:1.Phorbol-12-myristate-13-acetate induced THP-1 monocytes to differentiate into macrophages for 48 hours.The changes of THP-1 cells were analyzed from the following two aspects:(1)The morphological changes were observed under microscope.(2)The changes of CD 11b and CD 14 were measured by flow cytometry.2.CCK-8 assay was used to detect the effects of different concentrations of Hcy(0,25,50,100,250,500,1000,2000,5000 μM)on the viability of THP-1 macrophages.3.Detect the effect of different concentrations of Hcy(0,25,100,250,500 μM)on the autophagy of THP-1 macrophages:the number of autophagosomes was detected by electron microscope;autophagic vesicles were detected by MDC fluorescent dye method;the mRNA and protein expressions of LC3,p62 and Beclin-1 were detected by fluorescence quantitative PCR and Western blot,respectively.4.Western blot was used to detect the effects of Control,Hcy(500 μM),autophagy inhibitor 3-methyladenine(3-MA)(5 mM),autophagy inducer rapamycin(Rapa)(10 nM),Hcy+3-MA and Hcy+Rapa groups on LC3 protein levels in THP-1 macrophages.5.Western blot was used to detect the effects of different concentrations of Hcy(0,25,100,250,500 μM)on the levels of p-AMPK,p-mTOR,cytoplasmic and intracellular TFEB proteins in THP-1 macrophages.6.Western blot was used to detect the effects of Control,Hcy(500 μM),AMPK activator acadesine(AICAR)(500 μM),Hcy+AICAR group groups on the levels of LC3,p62,Beclin-1,p-AMPK,p-mTOR,cytoplasmic and intracellular TFEB protein of THP-1 macrophages.Results:1.After THP-1 cells were stimulated by PM A,the morphology of THP-1 cells showed the characteristics of macrophages under the microscope;Flow cytometry analysis showed that the expression levels of CD11b and CD14 were significantly increased(P<0.05).2.The results of CCK-8 experiment showed that different concentrations of Hcy had no significant effect on cell viability(P>0.05).3.Compared with the control group,Hcy down-regulated the protein expression levels of LC3 Ⅱ/Ⅰ and Beclin-1 in THP-1 macrophages in a dose-dependent manner,and up-regulated the protein level of p62(P<0.05).These are consistent with gene level expression.Hcy reduces the number and density of autophagosomes and intracellular autophagosomes in THP-1 macrophages.4.The autophagy inhibitor 3-MA can strengthen the inhibitory effect of Hcy on the autophagy of THP-1 macrophages(P<0.05),and the autophagy activator Rapa can weaken the inhibitory effect of Hcy on the autophagy of THP-1 macrophages(P<0.05).5.Hcy can down-regulate the expression levels of p-AMPK,nuclear TFEB and other proteins in a dose-dependent manner,and up-regulate the expression levels of p-mTOR and cytoplasmic TFEB proteins(P<0.05).6.AMPK activator AICAR can attenuate the inhibitory effect of Hcy on macrophage autophagy and activate AMPK/mTOR/TFEB signaling pathway(P<0.05).Conclusion:High Hcy reduces the autophagy level of THP-1 macrophages,which is achieved by inhibiting the AMPK-mTOR-TFEB signaling pathway.